The ability to produce ethanol from cellulosic feedstocks by fermentation on a commercial scale is a long sought goal. To be economical, the amount of ethanol produced must be sufficient to be worth the cost of preparation of the feedstock and processing of the final product. To produce sufficient amounts of ethanol requires a fermentation organism that is biologically efficient at ethanol production in comparison to production of other metabolic products. Efficiency is determined by yield and productivity, yield being expressed as a weight percentage of sugar feedstock (typically sucrose or glucose) converted into ethanol, and productivity being expressed as the maximum amount of ethanol that can be produced as a volume/volume percentage of the fermentation media before the fermentation ceases due to ethanol toxicity. In this regard, by far, the most efficient ethanol producing microorganism is the baker's yeast S. cerevisiae 
Efficient ethanol producing strains of S. cerevisiae are capable of converting 90-93% of a sugar based carbon source into ethanol on wt/wt basis and the ethanol can typically accumulate to 16-17% of the volume of the fermentation media. The sugar for commercial scale production of ethanol is conventionally obtained by extraction of sucrose from sugar beet or cane, or by hydrolysis of corn starch to produce glucose. Sucrose and corn starch, however, represent only a small fraction of the total sugars in plant material, most of which is contained in the β glucoside polymers cellulose and hemicellulose, the later being a branched polymer of the C6 sugars glucose and mannose and the C5 sugars xylose and arabinose.
There are several methods in the art for making hydrolysates of cellulose and hemicelluloses to produce feedstocks containing glucose, mannose, xylose and arabinose. For typical hydrolysates from corn stover, glucose represents 14.4%, mannose 0.9%, xylose 66.1% and arabinose 11.8% of the sugars. For typical hydrolysates of corn fiber glucose represents 48.6% xylose 25.2% and arabinose 17.6% of the sugars. Glucose and mannose are efficiently converted to ethanol during natural anerobic metabolism, however, S. cerevisiae, lacks the enzymatic machinery to convert the dominant sugar, xylose, into ethanol. To do so requires genetic engineering of S. cerevisiae to express metabolic enzymes that can convert xylose into xylulose phosphate—a C5 metabolite that is part of the pentose phosphate pathway, which ultimately produces intermediates that can enter the glycolytic pathway and be converted to ethanol during anaerobic fermentation. Normally within the pentose phosphate pathway, xylulose phosphate is derived from ribulose phosphate by the action of an epimerase, but in addition, S. cerevisiae contains the enzyme xylulokinase which can directly phosphorylate xylulose. Xylulose however, is a rare metabolite, and the level of expression of xylulokinase in S. cerevisiae is low. But more importantly, xylulokinase does not phosphorylate xylose and S. cerevisiae lacks the necessary enzymes to convert xylose to xyululose so is unable to utilize xylose as a carbon source without metabolic engineering.
There are two approaches to engineer S. cerevisiae to produce xylulose from xylose. The first represented by U.S. Pat. No 5,789,210 to Ho et al, is the XR-XD-XK three gene route, which is to overexpress xylulokinase (XK) simultaneously with an exogenous xylose reductase (XR) which reduces xylose to xylitol, and a xylitol dehydrogenase (XD), which oxidizes xylitol to xylulose. This approach, however, creates a redox imbalance in S. cerevisiae because xylose reductase utilizes NAD(H) as the reducing cofactor while xylitol dehydrogenase uses NADP+ as the oxidizing cofactor. This imbalance negatively affects the growth and productivity of S. cerevisiae shutting down efficient production of ethanol from xylose. One option to overcome this problem is to use a mutant xylose reductase that has a lower Km for NAD+ than NADP+ thereby restoring the redox balance as has been described for example by Petschacher B, et. al. (Biochem J2005, 385:75-83).
The second approach is the XI-XK two gene route, which is to overexpress xylulokinase along with an exogenous xylose isomerase (XI) which directly converts xylose to xylulose without reduction and subsequent oxidation. This approach is represented by: U.S. Pat. No. 7,622,284 and US Pat. Pub Nos: US20060216804, US20080261287. Genes from a variety of bacterial and fungal source of xylose isomerase share the common name xylA. Several species of xylA genes have been identified from bacterial and fungal sources and some, but not all, have been shown to be useful in producing ethanol from xylose simultaneously overexpressed with xylulokinase. Proposed bacterial sources for such xylA genes include Thermus thermophilus (U.S. Pat. No. 7,622,284), Bacteroides thetaiotaomnicron, (US20060216804, US20080261287) and Xanthamonus. Several fungal sources of xylA genes have also been proposed, including from Neocallimastix, Caecomyces, Piromyces, Orpinomyces, or Rumnomyces. (US20080261287). See also Curr Op Biotech 17:320 (2006). Of these, only the xylA genes from Piromyces (20080014620) Orpinomyces (Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S, Srivastava A, Fukuda H, Bisaria VS, Kondo A. Appl Microbiol Biotechnol. 2009 82(6):1067-78.) and Bacteroides thetaiotaomnicron (US20080261287) have been shown to improve ethanol production in S. cerevisiae when co-overexpressed with xylulokinase. See also FEMS Yeast Res 4:69, FEMS Yeast Res 5:399, FEMS Yeast Res 4:655, FEMS Yeast Res 5:925.
Although ethanol production from both the three gene approach and the two gene approach has been demonstrated, the ethanol yield form xylose remains lower than expected for strains only containing those features. To improve productivity requires further genetic manipulation, either by way of mutation, evolutionary selection or by further genetic engineering. For example, US20070082386 proposes that ethanol production from xylitol by either the two gene or three gene route could be improved by increasing expression of a xylose transporting enzyme and/or by overexpression of genes encoding enzymes of the pentose phosphate pathway. US20060234364 discloses that mutants having a deletion in an endogenous gre3 gene encoding a non specific aldol reductase could improve ethanol production from xylitol using the two gene approach. US20070155000, from a different perspective, teaches than ethanol production from xylose containing hydrolysates utilizing the two gene route could be improved by further selection for resistance against growth inhibitors such as furfural and hydroxymethyl furfural that are typically found in hydrolysates of lignocellulosic biomass.
There remains a need in the art to discover other xylose isomerase genes and other multi gene combinations to improve the efficiency of xylose utilization in S. cerevisiae for ethanol production. The disclosure that follows presents such alternatives in the form of a particular xylose isomerase xylA gene from Bacteroides fragilis and alternative three gene route that includes simultaneous overexpression of an xylose isomerase, xylitol dehydrogenase and xylulokinase activity, without the need for overexpressing xylose reductase.